Project Summary Vascular calcification is a hallmark of atherosclerotic cardiovascular diseases such as myocardial infarction and stroke, which are the leading causes of morbidity and mortality in the world. Although aortic calcification is a strong independent risk factor for cardiovascular disease, the genetic determinants of aortic calcification remain unknown and the molecular mechanisms of vascular calcification are incompletely elucidated. In a multi-cohort study with more than 9400 participants, we identified single nucleotide polymorphisms in the histone deacetylase 9 (HDAC9) locus that are associated with abdominal aortic calcification. In an in vitro model of calcification, our preliminary experiments demonstrated that inhibition of HDAC9 prevented the calcification of aortic vascular smooth muscle cells (VSMCs). In two different mouse models, we found that global HDAC9 deficiency protected against the development of vascular calcification and atherosclerosis. Based on our preliminary evidence, combining a human genome-wide association study, in vitro VSMC experiments, and a murine model of vascular calcification, we have identified HDAC9 as a novel activator of vascular calcification. The overall objective of this proposal is to understand the molecular mechanisms by which HDAC9 promotes vascular calcification and atherosclerosis. HDAC9 is a member of the Class IIa HDAC family, all of which possess both a deacetylase catalytic domain and a myocyte enhancer factor 2 (MEF2) binding site. First, using a series of VSMC functional assays, we propose to determine the role of HDAC9, and its downstream target MEF2, in promoting VSMC osteogenic phenotype switch and calcification. Second, to test the hypothesis that the influence of HDAC9 on vascular disease is exerted through its specific effects on VSMCs, we will determine if VSMC-specific deletion of HDAC9 in a murine model is sufficient to attenuate vascular calcification and atherosclerosis. Lastly, we have uncovered a novel interaction between HDAC9 and the long non-coding RNA MALAT1. We will determine if MALAT1 is required for the HDAC9- dependent effects on VSMC phenotype and on vascular calcification and atherosclerosis using a MALAT1 murine knockout model. The experiments proposed will provide important mechanistic insights into the function of HDAC9 in the vasculature and into the underlying molecular and cellular mechanisms of vascular calcification and atherosclerosis.